Yeast strains for improved ethanol production

ABSTRACT

Novel strains of yeast and methods for improved ethanol production utilizing the yeast strains are disclosed. In particular, the novel yeast strains  Saccharomyces cerevisae  YE1358 and YE1615 provide for increased fermentation temperature tolerance, as well as tolerance to increased levels of glucose and ethanol, and thereby provide increased ethanol production as compared to ethanol industry standard strains of  Saccharomyces cerevisae . The novel yeast strains also generate decreased residual glucose than the ethanol industry standard yeasts.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/232,673, filed Aug. 10, 2009, which is herebyincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

This invention relates to ethanol production processes and to isolatednovel yeast strains and use of the same for improved methods of ethanolproduction.

BACKGROUND OF THE INVENTION

Increasing demand for renewable energy sources, including ethanolproduction, has created commercial demand for the generation of ethanolfuel sources. The process to obtain ethanol generally involves thetransformation of plant matter, usually sugars, into a distilled pureethanol product, through either a wet or dry milling processes. Wetmilling methods include various soaking steps to soften grains andseparate the soluble starches (from the germ, fiber and proteincomponents) for further treatment before alcohol production methods. Drymilling methods are most commonly used for ethanol production, involvingthe grinding of whole plant matter into meal (without fractionating orseparating the various component parts of the starch, as is done withwet milling) and forming a slurry. Subsequently, the starch componentsof the slurry are converted into sugars, through the use of hightemperatures and enzymes, to effectuate the conversion. Upon generationof a sugar source from the plant matter, the conversion to ethanolrequires the use of an ethanol-producing microorganism, such as yeasts.

The ethanol-producing microorganism utilized in the fermentation processmost often involves yeast added to the mixture to convert sugars toalcohol, which is then distilled, purified, and denatured to generatethe ethanol end product. (U.S. Pat. No. 4,316,956). The majority of fuelethanol is produced by a few distinct strains of yeast. These commercialyeast strains require a high tolerance to both sugars and ethanol inorder to yield sufficient ethanol concentrations.

The various processes and method steps for producing ethanol are wellknown by those skilled in the art and are described in variousreferences. Ethanol production includes at least the following processesor method steps, or combinations of the same: milling, liquefaction,saccharification, fermentation and distillation. (See e.g., U.S. Pat.Nos. 5,231,017 and 4,316,956, WO 94/08027, WO 92/20777). One skilled inthe art can ascertain the process for production of ethanol, includingfor example, whether steps are carried out simultaneously.

Numerous improvements to the ethanol production process, including boththe wet and dry milling processes, have been achieved by those skilledin the art. For example: genetic modifications to ethanol-producingmicroorganisms (see e.g. Shi et al, J. Ind. Microbiol. Biotechnol.,36:139-47 (2009)); improving thermotolerance (see e.g. U.S. PublicationNo. 2005/0069998); and improved enzymatic processes and enzymes (seee.g. WO 2007/145912).

Despite improvements to methods of ethanol production, the process iscomplex and the significant commercial interest in ethanol creates acontinued need to further improve the production process, including theethanol-producing microorganisms utilized therein. Accordingly,improvements upon commercial yeasts strains are further necessary toenhance industrial production of ethanol. Ethanol-producingmicroorganisms, such as yeast, capable of improving ethanol production,as measured by the ability to produce increased ethanol yields, provideboth significant economic and industrial advantages. Even slightimprovements in ethanol production provide significant commercialbenefits. For example, it is estimated that an increased ethanol yieldof 1% (estimated as an additional 0.0271 gallons/bushel) generates acommercial value of $100 million dollars annually for the ethanolindustry (estimated as $2.00/gallon ethanol).

Accordingly, enhanced methods for ethanol production involving novel,isolated ethanol-producing microorganisms, such as yeast strains, willenhance production and profitability of industrial ethanol production.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated yeast strains collected fromdiverse environments and tested for improved ability to enhance ethanolproduction over industry standard yeast strains. Therefore, anembodiment of the invention is a biologically pure culture of YE1358,having NRRL Accession No. XXXX and a biologically pure culture ofYE1615, having NRRL Accession No. YYYY.

In one embodiment, isolated Saccharomyces cerevisae strains YE1358 andYE1615 enhance ethanol production, as demonstrated in the detaileddescription of the invention.

Embodiments of the invention further include methods for utilizingisolated yeast strains YE1358 and YE1615 for enhancing ethanolproduction through production of increased alcohol yields.

In another embodiment, isolated yeast strains YE1358 and YE1615 arethermotolerant. In a further embodiment, isolated yeast strains YE1358and YE1615 are tolerant to increased levels of glucose and ethanol, incomparison to the current industry standard Saccharomyces cerevisaestrains, resulting in increased ethanol production.

In yet another embodiment, ethanol production using yeast strains YE1358and YE1615, with improved temperature, glucose and ethanol toleranceresults in significantly decreased input costs for ethanol productionand increased ethanol production.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows genetic profiles of yeast strains YE1358, YE1615 andCommercial Yeast Strains.

FIGS. 2A-2D show HPLC analysis of yeast fermentation at varyingtemperatures.

FIGS. 3A-3C show the effect of temperature on conventional ethanolfermentation of selected isolated yeast strains.

FIGS. 4A-4B show the effect of temperature on conventional ethanolfermentation.

FIG. 5 shows ethanol production of selected yeasts.

FIGS. 6A-6B show HPLC analysis of yeast fermentations via theconventional starch hydrolysis method.

FIG. 7 shows ethanol yields from conventional starch hydrolysis assay ofselected yeasts.

FIGS. 8A-8B show HPLC analysis of selected yeast fermentations via theconventional starch hydrolysis method at various temperatures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments of this invention are not limited to particular yeaststrains or methods of improving ethanol production, which can vary asunderstood by skilled artisans. It is further to be understood that allterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting in any manner orscope. For example, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” can include pluralreferents unless the context clearly indicates otherwise. By way ofexample, “an element” means one or more elements. Further, all units,prefixes, and symbols may be denoted in their SI accepted form. Numericranges recited within the specification are inclusive of the numbersdefining the range and include each integer within the defined range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which embodiments of the invention pertain. Many methods andmaterials similar, modified, or equivalent to those described herein canbe used in the practice of the embodiments of the present inventionwithout undue experimentation, of which the preferred materials andmethods are described herein. In describing and claiming the embodimentsof the present invention, the headings provided are not limitations tothe embodiments of the invention and the following terminology will beused in accordance with the definitions set out below.

The term “alcohol (ethanol) tolerance” means an improved ability of aselected yeast strain to survive while maintaining function, such asproducing ethanol, in a medium having a higher concentration of alcohol,as compared to other yeast strains.

The term “Commercial Yeast Strains” means commercially available activedry alcohol yeasts, including for example, Fali (Fleischmann's Yeast)and Baker's ethanol yeast. These strains represent the industrystandard, traditionally demonstrating high alcohol tolerance, andthermotolerance, therefore producing maximum ethanol yields (up to 18%v/v (14.2% w/v), dependant on the fermentation methods). CommercialYeast Strains were utilized for baseline measurements as representativeof the current commercial microorganisms, preferably yeasts. TheCommercial Yeast Strains are specifically Saccharomyces, such as thestrain Saccharomyces cerevisiae.

The term “ethanol-producing microorganism” means any fermentingorganism, including yeast, capable of producing ethanol from saccharides(mono- or oligo-). The term, as used herein, is synonymous with“ethanologenic microorganisms” and “fermenting organisms”. A person ofordinary skill in the art can readily determine the effective amount ofethanol-producing microorganisms to be used in the methods of thepresent invention.

The term “fermentation” means the enzymatic and anaerobic breakdown oforganic substances by microorganisms; such as the process by whichsugars produce ethanol, carbon dioxide (waste product) and cellularenergy. Although yeast ferments ethanol without oxygen, it is understoodthat the process may occur in the presence of oxygen as an aerobicprocess. Methods of fermentation and other ethanol-producing methodsteps (including separation of end products, distillation, purificationand denaturation of ethanol) are well known in the art.

The term “glucose tolerance” means an improved ability of a selectedyeast strain to survive while maintaining function, such as producingethanol, in a medium having a higher concentration of glucose, ascompared to other yeast strains.

The term “milling” means any process of breaking down grains intosmaller particles. The term “dry milling” means any milling processwhereby components of dry grain (whole) are not separated, such as branand germ. The term “wet milling” means the milling process wherebycomponent parts of grain are first separated.

The term “plant material” refers to all or part of any plant (e.g.,cereal grains) and generally include a starch. For example, plantmaterial may include maize, sorghum, barley, wheat, rye, rice, etc. andmixtures of plant materials or plant material byproducts.

The term “saccharification” refers to the process of converting starchto smaller polysaccharides and monosaccharides, including for exampleglucose. Methods of saccharification are well known by those skilled inthe art to which the invention pertains.

The term “slurry” means an aqueous mixture of insoluble solids in themilling process for ethanol production.

The term “starch” means any complex polysaccharide carbohydrate plantmaterial. “Plant material” means any part of the plant selected from anyof the following, but not limited to the following, corn (Zea mays),wheat, sorghum, rice, millet, barley, potato, sugarcane and soybeans,and may include genetically modified varieties. The term “strain” meansany functional mutant or derivatives of the various yeast strainsdisclosed herein. For example, Saccharomyces cerevisae strain YE1358(NRRL Accession No. XXXX) and strain YE1615 (NRRL Accession No. YYYY),which retain the functional activity of improving ethanol yield asdescribed and defined by the methods and examples disclosed herein.Further, the term “functional mutant” means a yeast strain directly orindirectly obtained by genetic modification, of which may be achievedthrough any means, or using the referenced strains, and retaining theethanol production, thermotolerance, glucose tolerance and ethanoltolerance properties of at least one of strains YE1358 and YE1615.

The term “thermotolerance” means an improved ability of a selected yeaststrain to survive while maintaining function, such as producing ethanol,at increased temperatures, as compared to other yeast strains.

The term “yield” means generally the amount of end product, such asvarious types of ethanol (including for example fuel ethanol orindustrial ethanol), distillers dried grains and distiller's dried grainplus solubles, produced using the methods set forth in the presentinvention. Yield can refer to the concentration, volume, percentage ofincrease, and other means of measuring end products. The preferred endproduct yield measured with the invention is an alcohol product,preferably ethanol, which may be separated and/or purified according tomethods known to those of ordinary skill in the art. In some embodimentsof the invention, the yield of ethanol using yeast strains YE1358 andYE1615 will be greater than at least 10.8%, 11%, 11.2%, 11.4% 11.6%,11.8%, 12%, 12.2%, 12.4% 12.6%, 12.8%, 13%, 14.2%, 14.4% 14.6%, 14.8%,15%, 15.2%, 15.4% 15.6%, 15.8%, 16, 16.1%, 16.2%, 16.3%, 16.4% 16.5%,16.6%, 16.7%, 16.8%, 16.9%, 17% (v/v % ethanol production).

Isolated Yeast Strains

Isolated yeast strains Saccharomyces cerevisae YE1358 and YE1615 weredeposited on ______ with the Agricultural Research Service (ARS) CultureCollection, housed in the Microbial Genomics and Bioprocessing ResearchUnit of the National Center for Agricultural Utilization Research(NCAUR), under the Budapest Treaty provisions. The strains were giventhe indicated accession numbers: Saccharomyces cerevisae YE1358, NRRLAccession No. ______; and Saccharomyces cerevisae YE1615, NRRL AccessionNo. ______. The address of NCAUR is 1815 N. University Street, Peoria,Ill., 61604. The deposits will irrevocably and without restriction orcondition be available to the public upon issuance of a patent. Thesedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112. However, it should beunderstood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by government action. The deposit will be maintained withoutrestriction in the NRRL Depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the patent, whichever is longer, and will bereplaced if it ever becomes nonviable during that period.

Yeast strains YE1358 and YE1615 were isolated from inoculated andensiled whole plant corn silage with high levels of dry matter. Aftersignificant experimentation the strains YE1358 and YE1615 werediscovered from testing a collection of isolates. After isolation of thespecific strains, taxonomic studies were done to identify the strains.The strains YE1358 and YE1615 were identified as Saccharomyces cerevisaeand verified via DNA match reports (sequenced 323 base pairs from the D2region of the Fungal 28S rRNA gene) (MIDI Labs, Delaware). Further,genetic profiles were generated (FIG. 1) using a modified yeastmitochondrial DNA restriction analysis and demonstrating geneticdifferences between all strains. (Querol et al., 1992: Comparative Studyof Different Methods of Yeast Strain Characterization System, Appl.Microbiol. 15:439-46).

In embodiments of the invention, the enhanced ethanol-producing methodsproducing increased alcohol yields are accomplished by using the speciesSaccharomyces cerevisae as the ethanol-producing microorganism utilizedin the fermentation process. The novel, isolated yeast strains YE1358and YE1615, or compositions containing YE1358, YE1615 or closely relatedorganisms, are used to enhance ethanol production. Those of ordinaryskill in the art will know of various suitable alternative compositionsfor the isolated yeast strains YE1358 and YE1615, or compositionscontaining YE1358, YE1615 or closely related organisms using routineexperimentation.

The activity associated with the invention may be found in other strainsof Saccharomyces cerevisae and in other species of Saccharomyces andpossibly in other genera. This can be established by routineexperimentation, on the basis of the information provided hereinrelating to the invention.

Glucose and Ethanol Tolerance

In one embodiment, yeast strains YE1358 and YE1615 demonstrate superiorglucose tolerance over expected tolerance levels. (Elsevier, 1984,Yeasts: A Taxonomic Study, 88). Glucose concentrations up to 60% can beutilized as growth media for yeast strains YE1358 and YE1615 withoutrate-limiting growth. These glucose tolerance levels far exceed theindustrial ethanol fermentation standards of approximately 20% offermentable sugar.

In another embodiment, yeast strains YE1358 and YE1615 demonstrategrowth at ethanol concentrations up to 25%, exceeding the generallyaccepted ethanol concentration of 10-15%. Alcohol tolerance is abiologically limiting factor as yeast strains are inactivated andtherefore stop metabolizing and growing when exposed to the inhibitoryeffects of increased levels of ethanol. The ethanol tolerance of strainsYE1358 and YE1615 further exceeds even those ethanol tolerance levelsachieved in studies demonstrating the “coaching” of ethanol tolerance upto 23%. (Lyons, 1999, Alcohol Textbook 3rd Ed., vol. 1). In stillfurther embodiments of the invention, yeast strains YE1358 and YE1615demonstrate enhanced growth at both 15% and 20% ethanol concentrations(with a 60% glucose concentration), where Commercial Yeasts Strains wereunable to grow at 10% or greater ethanol concentrations (with a 60%glucose concentration), demonstrating an unexpected improvement over thealcohol tolerance of Commercial Yeast Strains.

In yet another embodiment, yeast strains YE1358 and YE1615 retain boththe alcohol and glucose tolerance described herein, simultaneously withits thermotolerance as set forth in this detailed description of theinvention. The improved tolerances of strains YE1358 and YE1615 furtherresult in increased yields of ethanol, according to percentage ofethanol yield.

Thermotolerance

According to the invention, yeast strains YE1358 and YE1615 are capableof producing ethanol at increased temperatures due to thermotolerance.The demonstrated thermotolerance of yeast strains YE1358 and YE1615allows at least maintained and increased production of ethanol atincreasing temperatures which are generally regarded as those exceedingacceptable temperatures for fermentation with yeasts. Strains YE1358 andYE1615 produce increased ethanol yields in comparison to one or moreCommercial Yeast Strains at temperature of at least above 31° C., 33°C., 35° C. and 37° C., contrary to the industry standard productionwhich experience decreased ethanol production as temperatures increase,due to the inhibitory effects of ethanol on yeast with increasedtemperatures. (D'Amore et al., Enzyme Microb. Technol., 11:411-16(1989)).

Maintained and increased production of ethanol yields at higherfermentation temperatures results in—decreased expenditures forcommercial ethanol plants. Thermotolerant yeasts provide numerousadvantages; including energy savings due to a reduced need for coolingfermenters and other compensatory means to ensure the yeasts remainactive. Therefore, it is economically and technically advantageous toferment ethanol at higher temperatures and with thermotolerant yeaststrains, including strains YE1358 and YE1615.

In yet another embodiment, yeast strains YE1358 and YE1615 retainthermotolerance simultaneously with ethanol and glucose tolerance, asset forth in this detailed description of the invention. These improvedtolerances of strains YE1358 and YE1615 further result in increasedyields of ethanol according to the present invention. These embodimentsprovide unexpected results of simultaneously maintaining ethanol,glucose, and thermotolerance, rather than showing a decrease in ethanolor glucose tolerance and, therefore, ethanol yields, as a result ofincreasing temperatures.

Ethanol Production

According to the invention, ethanol is the preferred end product fromthe claimed methods utilizing yeast strains YE1358 and YE1615. Inpreferred embodiments, the ethanol yield will exceed at least 10.8%,11%, 11.2%, 11.4% 11.6%, 11.8%, 12%, 12.2%, 12.4% 12.6%, 12.8%, 13%,14.2%, 14.4% 14.6%, 14.8%, 15%, 15.2%, 15.4% 15.6%, 15.8%, 16, 16.1%,16.2%, 16.3%, 16.4% 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17% (v/v %ethanol production). Ethanol yields generated from the claimed methodsof the present invention are identified by high-pressure liquidchromatography (HPLC) analysis. In the preferred embodiments of thepresent invention, increased ethanol production results in decreasedinput costs and increased economic value obtained from ethanolproduction.

The fermentation required for ethanol production is ongoing untilsufficient yields of ethanol are produced. Fermentation may be carriedout over extended periods of time, for example for up to 24 hours to 96hours. One of skill in the art may ascertain such variables of length offermentation to produce desired ethanol yields while utilizing the yeaststrains YE1358 and YE1615, according to the invention.

The invention provides methods for producing ethanol comprisingculturing a yeast strain of the invention in a medium comprising asaccharide, particularly an aqueous medium. Preferably, the yeast strainis capable of increasing ethanol yields when compared to a CommercialYeast Strain. More preferably, the yeast strain is selected from thegroup consisting of: Saccharomyces cerevisae strain YE1358 having NRRLAccession No. XXXX; Saccharomyces cerevisae strain YE1615 having NRRLAccession No. YYYY; and yeast strains, which are mutants or derivativesof strain YE1358 or strain YE1615, said mutants and derivativescomprising the ethanol production, thermotolerance, glucose tolerance,and ethanol tolerance properties of at least one of strains YE1358 andYE1615. The culturing is conducted at a temperature that is suitable forthe growth and multiplication of the yeast stain as disclosed elsewhereherein. In addition to at least one saccharide, the medium can containother components that promote the growth and multiplication of the yeaststrain of interest. Such other components include, for example, aminoacids, other nitrogen containing molecules, and one or more additionalsaccharides. Suitable media include, for example, media produced fromcorn, particularly corn grain.

The methods of the invention provide for increased ethanol productionwhen compared to the culturing of a Commercial Yeast Strain undersimilar conditions. Such methods can generate an increase in ethanolyield of at least about 1%, 1.5%, or 2%, when compared to ethanol yieldfrom a Commercial Yeast Strain cultured under the same conditions.

In another embodiment of the invention, increased ethanol production isfurther demonstrated by decreased residual glucose levels. Decreasedresidual glucose levels are desirable as an indicator of the extent ofethanol production. Ideal ethanol production methods would result in 0%remaining glucose and yield 100% ethanol. Utilizing 100% of the glucosesources enhances complete usage of the products and significantlydecreases input costs to ethanol production methods. Yeast strainsYE1358 and YE1615 have approached such ideal conditions, as demonstratedin the examples of the detailed description.

Yeast strains YE1358 and YE1615 yield lower residual glucose and higherethanol amounts, as a result of increased glucose conversion to ethanol.In preferred embodiments of the present invention, yeast strains YE1358and YE1615 yield residual glucose level less than 1%, and morepreferably, residual levels less than 0.88%, and still more preferably,residual levels less than 0.35%. Further, yeast strains YE1358 andYE1615 yield residual glucose level less than Commercial Yeast Strainsat temperatures greater than 31° C., more preferably at temperaturesgreater than 33° C., and even more preferably at temperatures greaterthan 35° C. or 37° C.

In yet another embodiment of the invention, increased ethanolproduction, as a result of decreased fermentation stress, is furtherdemonstrated by decreased glycerol levels. Glycerol is an indicator ofstress in the ethanol production methods, as it is a sugar sourcefailing to convert to ethanol, further resulting in decreased ethanolyields. In a preferred embodiment of the invention, yeast strains YE1358and YE1615 demonstrate low glycerol levels at increased temperatures ofat least above 31° C., above 33° C., above 35° C., and above 37° C. Ineven more preferred embodiments, fermentation stress with yeast strainsYE1358 and YE1615 is lower at increased temperatures.

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications to theembodiments of the invention to adapt it to various usages andconditions.

EXAMPLES Example 1 Glucose and Ethanol Tolerance

Yeast strains were initially screened for glucose tolerance using the96-well microtiter plate format. Yeast Nitrogen Base media (Difco) withvarying levels of glucose concentration (up to approximately 60%) wasutilized for the growth media and current Commercial Yeast Strains (Faliand Baker's yeast) served as baseline measurements. All platesdemonstrated sufficient growth at all glucose levels and CommercialYeast Strains grew at 60% glucose concentration.

Yeast strains were also screened for ethanol tolerance utilizing thesame Yeast Nitrogen Base utilized for the growth media, with 60% glucoseconcentration and varying levels of ethanol concentration (up toapproximately 25%). Commercial Yeast Strains did not grow at 10% orgreater ethanol concentration with the 60% glucose concentration. Yeaststrains Saccharomyces cerevisae YE1358 and YE1615 demonstrated eithergood growth or light growth at both 15% and 20% ethanol concentrationsand 60% glucose concentration.

Example 2 Ethanol Production

Yeast collections were further screened for ethanol production using acolorimetric kit and ethanol UV-methods (Boehringer Mannheim/R-BiopharmEnzymatic BioAnalysis/Food Analysis). Commercially realistic growthmedia was utilized for ethanol production screening; High Moisture Cornbroth (pH of 4.2) containing 2% glucose concentration and 1% 2M ureaconcentration. Multiple plates were grown at 30° C., 35° C., 40° C., and45° C. and subsequently 24 hour and 72 hour readings were recorded.Several yeast strains, including yeast strains Saccharomyces cerevisaeYE1358 and YE1615 isolates, produced an increased amount of ethanol incomparison to the Commercial Yeast Strain standards.

Example 3 Residual Glucose Levels

Assays for glycerol levels were also analyzed for the yeast strains,demonstrating low levels for all strains, including YE1358 and YE1615,indicative of low levels of fermentation stress. Strains YE1358 andYE1615 demonstrated lower residual glucose and higher ethanol amountsthan both Commercial Yeast Strains, indicating increased glucoseconversion to ethanol. None of the strains reached a 1% residual glucoselevel, indicative of incomplete fermentation. The HPLC results show adifference in the amount of byproduct acetic acid produced, indicating aheterogeneous metabolic conversion of glucose.

HPLC samples were collected at the final time point of 72 hours. (FIGS.2A-2D). The residual sugar was lowest at 33° C. and trending towardsincreasing at 35° C. and 37° C. Only yeast strains YE1358 and YE1615reached a residual glucose level below 1%, with 0.35% residual glucosefor YE1358 and 0.88% residual glucose for YE1615, respectively.Additionally, glycerol levels remained low for all strains at theincreased temperatures.

Example 4 Total Fermentables

The selected yeast isolates YE1615 and YE1358 were also twice analyzedby the Conventional High Total Fermentables Assay with the followingmethods. Corn grain substrate was dried and ground to 1 mm. The amountsof 25 g of ground corn, 73 g distilled water, 1 ml of 200 mM CaCl₂, and1 ml of 2 M urea was added to a 250 ml Erlenmeyer flask. A sir bar wasadded and the pH was adjusted to 6.5 using 1M NaOH. Additionally, 100 μlof a-amylase enzyme was added. The flasks were covered and placed inboiling water baths and stirred for 15 minutes. The flasks were thenplaced in a 70° C. water bath to cool. Once cooled, the pH was adjustedto 4.5 with HCl and 600 μl of glucoamylase was added. The flasks wereincubated while stirring at 50° C. for 1 hour. Then the flasks werecooled to 25° C. in a water bath and the sample yeast was added. Theflasks were stoppered with a rubber stopper with a needle to release theCO₂ produced. The initial weight was recorded for each flask which wereincubated at various temperatures. The flasks were stirred and theirweight loss recorded at specified time points.

The Conventional High Total Fermentables Assay was completed at 30° C.and 35° C. The initial ending time point was 48 hours (See, Table 1). At48 hours, Commercial Yeast Strains (both Baker's and Fali yeast)performed better at 30° C. in comparison to YE1615 and YE1358 yeaststrains. However, at 35° C. YE1615 performed slightly better than aCommercial Yeast Strain (Fali), where as YE1358 was slightly lower thanthe Commercial Yeast Strain. Overall weight loss (ethanol production)was greatest at the increased temperature. (FIGS. 3A-3C).

TABLE 1 24 hr Wt 48 hr wt 48 hr Sample ID Initial Wt 24 hr Wt Lost/g DMGal/BU 48 hr wt lost/g DM gal/BU Baker's (30° C.) 253.88 245.57 0.3722.817 245.22 0.3879 2.933142648 Fali (30° C.) 251.44 242.93 0.381 2.883242.71 0.3907 2.954761978 YE1358 (30° C.) 248.76 243.73 0.223 1.69240.26 0.38 2.873714085 YE1615 (30° C.) 250.99 244.91 0.271 2.053 242.410.3843 2.9057301 Bakers (35° C.) 251.75 244.45 0.328 2.48 244.02 0.34562.61342515 Fali (35° C.) 248.51 239.78 392 2.967 239.58 0.39963.022029206 YE 1358 (35° C.) 245.13 238.11 0.315 2.384 236.37 0.39232.96622042 YE1615 (35° C.) 251.81 244.35 0.336 2.538 242.87 0.40093.031514692 Baker's (30° C.) 246.99 238.54 0.378 2.861 238.24 0.39152.960424987 Fali (30° C.) 226.81 218.2 0.385 2.915 218.06 0.39132.95924129 YE1358 (30° C.) 243.74 237.78 0.266 2.01 235.27 0.3792.865742489 YE1615 (30° C.) 243.17 236.6 0.294 2.221 243.55 0.38622.92060602 Baker's (35° C.) 253.82 245.65 0.368 2.78 245.04 0.39352.975453082 Fali (35° C.) 252.24 243.43 0.397 3.002 243.31 0.40063.029299525 YE1358 (35° C.) 246.89 239.01 0.354 2.674 238.07 0.39432.981996586 YE 1615 (35° C.) 255.29 247.15 0.366 2.768 246.34 0.40093.031299159 Baker's (30° C.) 246.35 237.97 0.375 2.839 237.54 0.39452.983346404 Fali (30° C.) 244.07 235.52 0.384 2.901 235.25 0.39552.990362909 YE1358 (30° C.) 244.86 238.74 0.274 2.069 236.38 0.38022.874918462 YE1615 (30° C.) 253.89 246.78 0.318 2.407 245.23 0.38822.935493865 Baker's (35° C.) 261.74 253.61 0.366 2.769 253.06 0.38932.943532268 Fali (35° C.) 250.89 242.13 0.394 2.981 241.99 0.39883.015380293 YE1358 (35° C.) 248.35 240.46 0.354 2.6 239.5 0.3962.994649553 YE1615 (35° C.) 259.09 250.78 0.373 2.824 250.21 0.39743.004916923

The ending time was subsequently extended to 72 hours. At 72 hours, aCommercial Yeast Strain (Baker's yeast) performed slightly better thanthe isolated yeast strains at 30° C. However, at 35° C. both YE1615 andYE1358 yeast strains out performed both Commercial Yeast Strains.Additionally, at 37° C. YE1615 out performed both Commercial YeastStrains and YE1358 out performed Baker's yeast for ethanol production.Overall weight loss (indicative of ethanol production) was greatest atthe increased temperature of 37° C. (FIGS. 4A-4B; Table 2).

TABLE 2 Average Average Average 24 Hr. 48 Hr. 72 Hr. Sample ID Wt LossWt Loss Wt Loss Baker's (30° C.) 0.3763 0.3905 0.3953 Fali (30° C.)0.3795 0.3870 0.3890 YE1358 (30° C.) 0.2327 0.3830 0.3931 YE1615 (30°C.) 0.3184 0.3885 0.3941 Bakers (35° C.) 0.3572 0.3905 0.3969 Fali (35°C.) 0.3945 0.4030 0.4054 YE1358 (35° C.) 0.3361 0.4026 0.4073 YE1615(35° C.) 0.3225 0.4007 0.4083 Baker's (37° C.) 0.3213 0.3417 0.3426 Fali(37° C.) 0.3855 0.4023 0.4025 YE1358 (37° C.) 0.3277 0.3822 0.3834YE1615 (37° C.) 0.3424 0.4077 0.4116 Bakers Control 0.3060 0.3060 0.3323(37° C.)

The amounts of ethanol production (both w/v % and v/v %) at each of 24hours, 48 hours and 72 hours were calculated. (Table 3). As supported bythe weight loss data, both YE1615 and YE1358 yeast strains demonstratedenhanced ethanol production over at least one or both of the analyzedCommercial Yeast Strains at 48 hours at temperatures of 35° C. and 37°C. and 72 hours at temperatures of 30° C., 35° C. and 37° C. Alcoholproductions of up to 16.57 v/v % are shown. (Table 3). The demonstratedethanol concentration from fermentation of YE1615 and YE1358 yeaststrains is a function of initial solids loading, wherein the methods ofthe experiments utilized low initial solids loading (25% as-is weight).Accordingly, the generated ethanol concentration is also lower inrespect to commercial or industry standard measurements of ethanolproduction, as demonstrated by the lower percentages of ethanolproduction achieved by the tested Commercial Yeast Strains (both Faliand Baker's yeast). The control standards provided by the CommercialYeast Strains were demonstrated from fermentation at the same initialsolids loading (rather than defined conditions such as an initial solidsloading as are necessary to generate ethanol production as great as 18%v/v as is often cited by Commercial Yeast Strains). Accordingly, Table 3shows the differences between the tested strains utilizing the same lowinitial solids loading.

TABLE 3 Average Average Average Average Average Average 24 Hr. 24 Hr. 48Hr. 48 Hr. 72 Hr. 72 Hr. EtOH EtOH EtOH EtOH EtOH EtOH Sample ID v/v %*w/v %* v/v % w/v % v/v % w/v % Baker's 15.38 12.13 15.86 12.51 16.0212.64 (30° C.) Fali 15.49 12.22 15.74 12.42 15.81 12.47 (30° C.) YE135810.15 8.01 15.61 12.32 15.95 12.58 (30° C.) YE1615 13.35 10.53 15.7912.46 15.98 12.61 (30° C.) Bakers 14.72 11.61 15.86 12.51 16.08 12.69(35° C.) Fali 16.00 12.62 16.28 12.84 16.36 12.91 (35° C.) YE1358 13.9811.03 16.27 12.84 16.42 12.96 (35° C.) YE1615 13.50 10.65 16.20 12.7816.46 12.99 (35° C.) Baker's 13.45 10.61 14.18 11.19 14.21 11.21 (37°C.) Fali 15.69 12.38 16.26 12.83 16.26 12.83 (37° C.) YE1358 13.68 10.7915.58 12.29 15.62 12.32 (37° C.) YE1615 14.20 11.20 16.44 12.97 16.5713.07 (37° C.) Bakers Control 12.90 10.18 13.72 10.83 13.85 10.93 (37°C.)

HPLC samples taken at 48 hours showed that the isolated yeast strainYE1615 produced the most ethanol at both 30° C. and 35° C. (FIG. 5). OneCommercial Yeast Strain (Fali) had slightly lower residual glucose thanthe other Commercial Yeast Strain (Baker's), however, both yeast strainsYE1615 and YE1358 had residual glucose levels below 1%. Althoughglycerol levels were low for both yeast strains YE1615 and YE1358, thestrain YE1615 demonstrated the lowest level of residual glucose. (FIGS.6A-6B).

HPLC samples taken at 72 hours showed the YE1615 yeast strain producedslightly less ethanol then one Commercial Yeast Strain (Fali) at 30° C.(FIG. 7). However, the YE1615 yeast strain still had the lowest levelsof residual glucose and glycerol at 30° C., despite all residual glucoselevels below 1%. (FIG. 8A). Additionally, glycerol levels were low forall tested yeasts. At 35° C. the YE1615 yeast strain produced the mostethanol (FIG. 7) and had the lowest levels of residual glucose andglycerol. (FIG. 8B).

The various modifications of the embodiments of the invention, inaddition to those shown and described herein, will be apparent to thoseskilled in the art from the foregoing description and Examples. Suchmodifications are also intended to fall within the spirit and scope ofthe appended claims.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1. An isolated Saccharomyces cerevisae yeast strain selected from thegroup consisting of: Saccharomyces cerevisae strain YE1358 having NRRLAccession No. XXXX; Saccharomyces cerevisae strain YE1615 having NRRLAccession No. YYYY; and yeast strains, which are mutants or derivativesof strain YE1358 or strain YE1615, said mutants and derivativescomprising the ethanol production, thermotolerance, glucose tolerance,and ethanol tolerance properties of at least one of strains YE1358 andYE1615.
 2. The yeast strain of claim 1, wherein the yeast strain isSaccharomyces cerevisae strain YE1358 having NRRL Accession No. XXXX. 3.The isolated yeast strain of claim 1, wherein the yeast strain isSaccharomyces cerevisae strain YE1615 having NRRL Accession No. YYYY. 4.A method for producing ethanol comprising culturing a yeast strain in amedium comprising a saccharide, wherein the yeast strain is selectedfrom the group consisting of: Saccharomyces cerevisae strain YE1358having NRRL Accession No. XXXX; Saccharomyces cerevisae strain YE1615having NRRL Accession No. YYYY; and yeast strains, which are mutants orderivatives of strain YE1358 or strain YE1615, said mutants andderivatives comprising the ethanol production, thermotolerance, glucosetolerance, and ethanol tolerance properties of at least one of strainsYE1358 and YE1615.
 5. The method of claim 4, wherein the yeast strain iscapable of increasing ethanol yields when compared to a Commercial YeastStrain.
 6. The method of claim 4, wherein said yeast strain isSaccharomyces cerevisae strain YE1358 or Saccharomyces cerevisae strainYE1615.
 7. The method of claim 4, wherein the temperature is above 31°C.
 8. The method of claim 4, wherein the temperature is above 33° C. 9.The method of claim 4, wherein the temperature is above 35° C.
 10. Themethod of claim 4, wherein the temperature is above 37° C.
 11. Themethod of claim 9, wherein said increased ethanol production generates ayield increase of at least about 1% compared to a Commercial YeastStrain.
 12. The method of claim 8, wherein said increased ethanolproduction generates a yield increase of at least about 1.5% compared toa Commercial Yeast Strain.
 13. The method of claim 4, wherein there is aresidual glucose level less than 1%.
 14. The method of claim 13, whereinthe residual glucose level for YE1358 is less than or equal to about0.35% and said residual glucose level for YE1615 is less than or equalto about 0.88%.
 15. The method of claim 4, wherein said yeast strain isglucose tolerant in glucose concentrations up to at least about 60%. 16.The method of claim 4, wherein said yeast strain is ethanol tolerant inethanol concentrations up to at least about 25%.
 17. The method of claim16, wherein said yeast strain are ethanol tolerant in ethanolconcentrations up to at least about 20% and glucose concentrations up toat least 60%.
 18. The method of claim 4 further comprising the stepsselected from the group consisting of milling, liquefaction,saccharification, distillation, ethanol extraction and combinations ofthe same.
 19. A method for increasing ethanol production by fermentationcomprising culturing a yeast strain in a medium comprising a saccharide,wherein the yeast strain is selected from the group consisting of:Saccharomyces cerevisae strain YE1358 having NRRL Accession No. XXXX;Saccharomyces cerevisae strain YE1615 having NRRL Accession No. YYYY;and yeast strains, which are mutants or derivatives of strain YE1358 orstrain YE1615, said mutants and derivatives comprising the ethanolproduction, thermotolerance, glucose tolerance, and ethanol toleranceproperties of at least one of strains YE1358 and YE1615.
 20. The methodof claim 19, wherein ethanol production is increased at least about 1%when compared to fermentation with a Commercial Yeast Strain.